Molecular and cytogenetic studies strongly implicate the plasticity of the cancer cell genome in malignant progression. Gene amplification is one prominent example of this genetic flexibility which is peculiar to malignant cells. The overexpression of proto oncogenes mediated by gene amplification has now been described in a variety of tumors of human and rodent origin, and in some cases it has been shown how such overproduction can engender increased invasiveness, ability to evade immune surveillance, etc. In addition, there is now ample documentation in human tumors in vivo that gene amplification can lead to resistance to a variety of antineoplastic agents alone, or in combination. Therefore, understanding the mechanisms of gene amplification could lead to a better understanding of how the control mechanisms which insure genetic stability in normal cells are abrogated in cancer cells, and the types of conditions which favor the occurrence of the amplification process. Such information could lead to new strategies of drug delivery to avoid development of resistance by this mechanism. This proposal presents experiments to investigate the nature of the earliest molecular products produced by gene amplification. Specifically, it will be tested whether submicroscopic, autonomously replicating circular elements are commonly generated as a first step. This hypothesis is formulated on the basis of previous work from this laboratory which demonstrated that such elements can be precursors of minute chromosomes. The structure of such elements will be analyzed using physical and molecular cloning methods designed to study genetic regions spanning hundreds of kilobases, and the hypothesis that such elements comprise functional replication domains will be tested. A replication origin present in one such element described previously in this lab will be isolated and analyzed in order to deduce how its structure contributes to regulated replication. Finally, a gene transfer method is proposed for identifying regions of the human genome associated with the capacity for high frequency gene amplication. By analogy with previous results from this lab, this approach could enable isolation of a human replication origin for further molecular characterization.